The method defined above includes chromatographic procedures that use monolithic or particle adsorbents in the form of packed or fluidised beds, and batch-wise procedures that typically include only particle adsorbents. Monolithic adsorbents include porous membranes, porous plugs and also tube walls and other forms of integral matrices. The purpose of the procedures may be to purify the substance carrying the charge, in which case the substance becomes bound to the adsorbent during step (i), and, if necessary, is further purified subsequent to desorption from the adsorbent. Another purpose is to remove the substance from liquid (I) because it is an undesired component therein. In this latter case, the liquid may be further processed after step (i). In both cases and if so desired, the adsorbent may be reused after desorption of the bound substance.
Other uses are assay procedures involving determination of either the substance carrying the charge or of a substance remaining in liquid (I).
There are a number of publications, which describe adsorbents that are functionalized with more than one kind of ligand.                WO 9600735, WO 9609116 and U.S. Pat. No. 5,652,348 (Burton et al) disclose separation methods and media based on hydrophobic interaction. In one embodiment the media may contain both ionizable and non-ionizable ligands. The main idea is that loading is done under conditions promoting hydrophobic interaction (neutral hydrocarbon ligands) and desorption by a pH switch in order to charge ligands with an opposite charge compared to the adsorbed protein (repulsion). Thus, WO 00/69872 utilises two ligands on the matrix, one of which is interacting with a nucleic acid during adsorption and the other one of which is utilised for desorption thereof by repulsion of the nucleic acid.        Burton et al., Biotechnology and Bioengineering 56(1) (1997) 45–55 describe attempts to purify chymosin on adsorbents comprising                    (a) aromatic hydrocarbon ligands that are chargeable but essentially uncharged during adsorption (secondary amine/ammonium), or            (b) an unchargeable aromatic ligand plus a separate cation-exchange ligand corresponding to the unreacted spacer (—COO−/COOH) (which has been used to introduce the aromatic ligand).                        Issaq et al., J. Liq. Chromatog. 11(14) (1988) 2851–2861; Floyd et al., Anal. Biochem. 154 (1986) 570–577; and Buzewski et al J. Liq. Chrom. & Rel. Technol. 20(15) (1997) 2313–2325 describe chromatographic properties of silica particles derivatized with two kinds of ligands (an ion-exchange ligand and a hydrophobic (alkyl) interaction ligand)        Teichberg, J. Chromatog. 510 (1990) 49–57 describes affinity repulsion chromatography in which a positively charged ligand is interacting with a neutral affinity ligand on an adsorbent that in addition also carries a positively charged repulsion ligand.        WO 9839094 (Amersham Pharmacia Biotech AB) and WO 9839364 (Amersham Pharmacia Biotech AB) disclose as one embodiment beads in which there is one kind of charged ligands in a surface layer while the interior of the beads is functionalised with ligands of the opposite charge. The beads are suggested for the adsorption of biomolecules.        It is known that the introduction of affinity ligands on separation matrices often introduces more than one kind of groups and/or residual groups due to inefficiencies in the coupling reaction. Reaction of N,N-diethyl aminoethyl chloride with polysaccharide matrices, for instance, typically introduces (a) ligands only containing one tertiary ammonium group together with (b) ligands containing both tertiary and quaternary ammonium groups. To our knowledge unusual high breakthrough capacities at ion-exchange conditions comprising high salt concentrations have never been reported for this type of conventional ion-exchangers. Compare Burton et al., Biotechnology and Bioengineering 56(1) (1997) 45–55        